RESUMO
In vertebrates, the nuclear pore complex (NPC), the gate for transport of macromolecules between the nucleus and the cytoplasm, consists of approximately 30 different nucleoporins (Nups). The Nup and SUMO E3-ligase Nup358/RanBP2 are the major components of the cytoplasmic filaments of the NPC. In this study, we perform a structure-function analysis of Nup358 and describe its role in nuclear import of specific proteins. In a screen for nuclear proteins that accumulate in the cytoplasm upon Nup358 depletion, we identified proteins that were able to interact with Nup358 in a receptor-independent manner. These included the importin α/ß-cargo DBC-1 (deleted in breast cancer 1) and DMAP-1 (DNA methyltransferase 1 associated protein 1). Strikingly, a short N-terminal fragment of Nup358 was sufficient to promote import of DBC-1, whereas DMAP-1 required a larger portion of Nup358 for stimulated import. Neither the interaction of RanGAP with Nup358 nor its SUMO-E3 ligase activity was required for nuclear import of all tested cargos. Together, Nup358 functions as a cargo- and receptor-specific assembly platform, increasing the efficiency of nuclear import of proteins through various mechanisms.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Carioferinas/metabolismo , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Transporte Proteico/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Núcleo Celular/metabolismo , Citoplasma/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Células HeLa , Humanos , Carioferinas/genética , Chaperonas Moleculares/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Membrana Nuclear/metabolismo , Sinais de Localização Nuclear/genética , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Mutação Puntual/fisiologia , Ligação Proteica/fisiologia , Domínios e Motivos de Interação entre Proteínas/fisiologia , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Deleção de Sequência/fisiologia , Transfecção , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Fatores Estimuladores Upstream/genética , Fatores Estimuladores Upstream/metabolismo , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismo , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismoRESUMO
The signaling molecule 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) has been described as the "anti-inflammatory prostaglandin." Here we show that substrates of the nuclear export receptor CRM1 accumulate in the nucleus in the presence of 15d-PGJ(2), identifying this prostaglandin as a regulator of CRM1-dependent nuclear protein export that can be produced endogenously. Like leptomycin B (LMB), an established fungal CRM1-inhibitor, 15d-PGJ(2) reacts with a conserved cysteine residue in the CRM1 sequence. This covalent modification prevents the formation of nuclear export complexes. Cells that are transfected with mutant CRM1 (C528S) are resistant to the inhibitory effects of LMB and 15d-PGJ(2), demonstrating that the same single amino acid is targeted by the two compounds. Inhibition of the CRM1 pathway by endogenously produced prostaglandin and/or exogenously applied 15d-PGJ(2) may contribute to its anti-inflammatory, anti-proliferative, and anti-viral effects.
Assuntos
Anti-Inflamatórios/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Carioferinas/metabolismo , Prostaglandina D2/análogos & derivados , Receptores Citoplasmáticos e Nucleares/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Sequência de Aminoácidos , Anti-Inflamatórios/química , Sobrevivência Celular/efeitos dos fármacos , Cisteína/metabolismo , Ácidos Graxos Insaturados/farmacologia , Células HeLa , Humanos , Carioferinas/química , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Prostaglandina D2/química , Prostaglandina D2/farmacologia , Receptores Citoplasmáticos e Nucleares/química , Proteína Exportina 1RESUMO
c-Fos, a component of the transcription factor AP-1, is rapidly imported into the nucleus after translation. We established an in vitro system using digitonin-permeabilized cells to analyze nuclear import of c-Fos in detail. Two import receptors of the importin beta superfamily, importin beta itself and transportin, promote import of c-Fos in vitro. Under conditions where importin beta-dependent transport was blocked, c-Fos still accumulated in the nucleus in the presence of cytosol. Inhibition of the transportin-dependent pathway, in contrast, abolished import of c-Fos. Furthermore, c-Fos mutants that interact with transportin but not with importin beta were efficiently imported in the presence of cytosol. Hence, transportin appears to be the predominant import receptor for c-Fos. A detailed biochemical characterization revealed that the interaction of transportin with c-Fos is distinct from the interaction with its established import cargoes, the M9 sequence of heterogeneous nuclear ribonucleoprotein A1 or the nuclear localization sequence of some basic proteins. Likewise, the binding sites on importin beta for its classic import cargo and for c-Fos can be separated. In summary, c-Fos employs a novel mode of receptor-cargo interaction. Hence, transportin may be as versatile as importin beta in recognizing different nuclear import cargoes.